Note that lysozyme is ~14 kDa so it will run close to my protein on a gel! Kathleen says if it is going to be a problem, freeze-thaw only should work reasonably well for this test. It is hard to sonicate small volumes. Could also try a commercial "mild lysis" reagent, although people in the Sauer lab have had varied success with these.

Save 13 μL resuspended pellet to load on a gel. (This is the insoluble fraction).

Notes

The amount of material you load from the supernatant and pellet should add up to the total protein so that you are comparing equivalent amounts.

Tom pointed out that a confounding factor is how well my cells lyse in the "native" versus "denatured" conditions. If my cells don't lyse in the native conditions then my protein will end up in the pellet even if they are soluble. However, it turns out that the "native" lysis protocol above seems to be more efficient than typical "denaturing" lysis (add denaturing lysis buffer and incubate with shaking for 1 hr at room temperature). Thus, the freeze thaw method may result in more complete cell lysis.